Journal: Immunity

Article Title: Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2

doi: 10.1016/j.immuni.2018.10.011

Figure Lengend Snippet: (A-B) RNA-seq analysis of gene expression in bone marrow macrophages (BMMs) stimulated for 6 h with: IL-4 (M(IL-4)) or PGE2 (M(−)+PGE2)(A) or M(IL-4) or M(IL-4)+PGE2 (B), compared to no stimulus controls (M(−)) (A,B). (C) Expression of CD301 and RELMα proteins, as measured by flow cytometry, in BMMs stimulated for 24 h with indicated treatments. MFI = mean fluorescence intensity. (D) Western blot of phosphorylated STAT6 (P-STAT6) at indicated times in BMMs treated as indicated. ACTB, actin loading control. (E-F) Extracellular flux analysis (EFA) of oxygen consumption rates (OCR) (E) or extracellular acidification rates (ECAR) (F) at 24 hr post stimulation. For EFA, BMMs were sequentially treated with oligomycin (Oligo), FCCP and rotenone plus antimycin A (R/A) as indicated. (G) 13C-Glucose LC-MS trace into TCA cycle metabolites from cells stimulated for 24 h with indicated treatments. (H-K) ATP concentration (H), cytoplasmic ROS (I), mitochondrial ROS (J), and Δψm as measured by TMRM incorporation (K), in BMMs stimulated as indicated for 24 h. (A-B) Significant (adjusted p value < 0.1) > 2 fold up or down regulated genes based on 3 biological replicates denoted by *. (C) Mean ± SEM from 5 biological replicates (p***<0.0005, p****<0.0001). (G) Mean ± SEM from 3 biological replicates, normalized to M(−) (p**<0.005). (H-K) Mean ± SEM from 5 biological replicates (p*<0.05, p***<0.0005, p****<0.0001). Data are representative of 2 (D) or 3 (C, E, F, H-K) independent experiments. See also Figure S1.

Article Snippet: Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (Qiagen), which makes predictions using pair-wise comparisons between conditions, incorporating in the analysis the statistically significant changes in expression within each of those comparisons. . Flow cytometry Used fluorochrome-conjugate monoclonal antibodies included: CD301 (Milteny Biotech, clone: REA687), CD11b (Biolegend, clone: M1/70), F4/80 (Biozol, clone: BM8), TIM4 (BioLegend, clone: F31–5G3), CD45.1 (BioLegend, clone: A20).

Techniques: RNA Sequencing, Gene Expression, Expressing, Flow Cytometry, Fluorescence, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets

doi: 10.1084/jem.20190723

Figure Lengend Snippet: Desialylated platelets are phagocytosed by Kupffer cells in vitro, Mac1 is dispensable for the clearance of desialylated platelets, Kupffer cells express Asgr1 and MGL1/2, but Asgr1-deficiency alone has no effect on platelet count . (A) Mice were treated with 50 mU sialidase i.v., and the amount of platelet accumulation in the liver at 30 min and 24 h after treatment was quantified as the CD49b-positive area from images obtained using SD-IVM. Data represent means ± SEM ( n ≥ 3 mice per group). (B) Kupffer cells were incubated with platelets in vitro, and phagocytosis was observed using an IncuCyte incubation microscope. Kupffer cells and platelets were labeled using F4/80 antibodies and CellTracker Red dye, respectively. Representative images showing Kupffer cell platelet co-culture at 0 h (upper panel) and at 5 h (lower panel). (C) Quantification of phagocytosis (Kupffer cells with red platelet signal inside) over time. (D) Wild-type and Mac-1 −/− mice were i.v. injected with 50 mU sialidase, and platelet accumulation in the liver was quantified from SD-IVM videos. n = 5 mice per group. (E) Kupffer cells were isolated from mouse liver, and ASGR1 expression was analyzed using flow cytometry. Representative flow plot showing ASGR1 signal versus fluorescence minus one (FMO). (F) Platelet count in blood from wild-type and Asgr1 −/− mice was quantified using flow cytometry. Data represent means ± SEM ( n = 5 mice per group). (G) Kupffer cells were isolated from mouse liver and MGL expression analyzed using flow cytometry. Representative flow plot showing MGL signal versus fluorescence minus one (FMO). (H) Washed human platelets and desialylated washed human platelets were incubated with fluorescently labeled recombinant human MGL (hMGL), and binding was quantified using flow cytometry. Representative flow plot. (I) Quantification of the binding of hMGL to control and desialylated human platelets from healthy human volunteers. Data represent means ± SEM ( n = 3). Unpaired two-tailed t test (A, F, and I) was used to determine statistical significance. ***, P < 0.001; ns, not significant. Results from all experiments shown are representative of at least three independent experiments.

Article Snippet: The MGL1/2 (CD301a/b) and AMR (ASGR1, clone #352803) antibodies, as well as recombinant human MGL (CD301/CLEC10A), were obtained from R&D Systems and labeled with Alexa647 dye using a Microscale Protein Labeling Kit (Thermo Fisher Scientific).

Techniques: In Vitro, Incubation, Microscopy, Labeling, Co-Culture Assay, Injection, Isolation, Expressing, Flow Cytometry, Fluorescence, Recombinant, Binding Assay, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets

doi: 10.1084/jem.20190723

Figure Lengend Snippet: A collaboration between MGL and AMR facilitates binding of desialylated platelets to Kupffer cells. (A) SD-IVM was performed on the liver of control mice, mice treated with ASF, Asgr1 −/− mice, wild-type mice treated with MGL1/2 blocking antibody (Ab), and Asgr1 −/− mice treated with MGL1/2 blocking antibody after injection of 50 mU sialidase. Kupffer cells and platelets were labeled using anti-F4/80 and anti-CD49b antibodies, respectively. Representative images taken at the indicated time points. Scale bars are 70 µm, except for the WT+MGL1/2 antibody images, where scale bars are 100 µm. (B) Quantification of platelet accumulation shown in A with n ≥ 4 mice per group, normalized to control mice values. Results from all experiments shown are representative of at least three independent experiments.

Article Snippet: The MGL1/2 (CD301a/b) and AMR (ASGR1, clone #352803) antibodies, as well as recombinant human MGL (CD301/CLEC10A), were obtained from R&D Systems and labeled with Alexa647 dye using a Microscale Protein Labeling Kit (Thermo Fisher Scientific).

Techniques: Binding Assay, Control, Blocking Assay, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets

doi: 10.1084/jem.20190723

Figure Lengend Snippet: Old platelets show significantly reduced hemostatic capacity. (A and B) Flow cytometric analysis of platelets from mice 5 d after Kupffer cell depletion shows increased P-selectin exposure in resting conditions, but decreased P-selectin exposure after stimulation with thrombin (Thr) compared with control platelets (A) and significantly reduced αIIbβ3 integrin activation (JON/A-PE binding; B) after stimulation with thrombin. Data represent means ± SEM ( n = 5 mice per group). (C) Representative aggregation traces of platelets isolated from control mice or mice 5 d after Kupffer cell depletion stimulated with 0.25 U/ml thrombin. (D and E) Maximum aggregation (A max ; D) and aggregation at the end time point (A end ; E) of platelets isolated from control mice or mice 5 d after Kupffer cell depletion. Data represent means ± SEM ( n = 3 mice per group). Control mice and mice 5 d after Kupffer cell depletion were subjected to the tail bleeding time assay. (F) Time until bleeding cessation; values >20 min indicate that bleeding did not stop within the 20-min observation period of the experiment. (G) Quantification of the amount of blood loss. Data represent means ± SEM ( n = 10 mice per group). (H) SD-IVM was performed on the liver of control mice after injection of cold-stored platelets. Kupffer cells and platelets were labeled using anti-F4/80 and CellTracker Red dye, respectively. Representative image taken at 30 min after injection. Scale bar is 70 µm. (I) Quantification of the accumulation of cold-stored platelets in wild-type and Asgr1 −/− mice as well as Asgr1 −/− mice treated with MGL-blocking antibody. n ≥ 4 mice per group, normalized to control mice values. Unpaired two-tailed t test (A, B, D, E, and G) was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results from all experiments shown are representative of at least three independent experiments.

Article Snippet: The MGL1/2 (CD301a/b) and AMR (ASGR1, clone #352803) antibodies, as well as recombinant human MGL (CD301/CLEC10A), were obtained from R&D Systems and labeled with Alexa647 dye using a Microscale Protein Labeling Kit (Thermo Fisher Scientific).

Techniques: Control, Activation Assay, Binding Assay, Isolation, Injection, Labeling, Blocking Assay, Two Tailed Test

Journal: JCI Insight

Article Title: Leukotriene B 4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models

doi: 10.1172/jci.insight.96902

Figure Lengend Snippet: BMDMs from aged BLT1-WT (filled circles) and BLT1-KO (open circles) mice (>20 weeks old) were polarized to M1 and M2 macrophages, and BLT1 expression was examined by quantitative PCR (A) and Flow cytometry analysis (B). M0 denotes a sample of nonpolarized BMDMs. RAW264.7 (C) and THP-1 (D) cells were polarized to M0 (cross plots), M1 (open squares), and M2 (filled circles) macrophages, and expression of BLT1, PD-L2 (an M2 marker), and CD80 (an M1 marker) was examined by flow cytometry. The y axis shows the MFI relative to that of an isotype control (mouse IgG1). MFI, mean fluorescence intensity. n = 3 per group. (E–H) FACS analysis of M2 macrophages isolated from the laser-injured eyes of aged WT mice (>20 weeks old) as described in Figure 3, C and D. CD206+ M2 macrophages, CD11bhiF4/80hiCD206+; CD206+BLT1+M2 macrophages, CD11bhiF4/80hiCD206+BLT1+. n = 3–4 per group. (I) FACS analysis of M2 markers on the ocular-infiltrating BLT1+ and BLT1– macrophages on day 5 after laser injury from aged WT mice. CD206, CD301, and PD-L2 are specific surface markers of M2 macrophages. Cells were stained with anti-CD206, anti-CD301, and anti–PD-L2 mAbs (black outlines) or isotype controls (gray filled histograms). Overlays show the expression level of M2 markers on BLT1+ (black outlines) and BLT1– (black dotted lines) macrophages. These macrophages were gated as the CD45+F4/80hiCD11bhi population. (J and K) FACS analysis of BLT1 expression on the ocular-infiltrating CD206+ M2 macrophages on day 3 after laser injury from young or aged WT mice. n = 3 per group. (A–D) **P < 0.01; ***P < 0.005; ****P < 0.001 versus 0 hours (2-way ANOVA with Bonferroni’s post hoc test). (E–H) *P < 0.05; **P < 0.01 versus day 0 (1-way ANOVA with Dunnett’s post hoc test). (K) **P < 0.01 (Student’s t test). Results are representative of at least 2 independent experiments (A–I).

Article Snippet: Cells were then incubated with mouse Fc blocker, followed by staining with anti–CD45-FITC (2.5 μg/ml; clone 30-F11; Becton Dickinson), anti–Ly6G-PE/Cy7 (2.5 μg/ml; clone 1A8), anti–Ly6C-APC/Cy7 (2.5 μg/ml; clone HK1.4), anti–F4/80-Brilliant Violet 421 (2.5 μg/ml; clone BM8), anti–CD11b-Brilliant Violet 510 (5 μg/ml; clone M1/70), and anti–mouse BLT1-APC (5 μg/ml; clone 7A8; generated in-house) Abs in addition to 2.5 μg/ml anti–PD-L2 (clone TY25; BioLegend), 10 μg/ml anti-CD301 (clone ER-MP23; Bio-Rad), 10 μg/ml anti-CD206 (clone MR5D3; BioLegend) biotin-conjugated Abs or isotype controls.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Marker, Fluorescence, Isolation, Staining

Journal: Cell

Article Title: A blood atlas of COVID-19 defines hallmarks of disease severity and specificity

doi: 10.1016/j.cell.2022.01.012

Figure Lengend Snippet:

Article Snippet: CD301 (H037G3)-167Er , Fluidigm , Cat# 3167015B, RRID: AB_2892694.

Techniques: Mass Cytometry, Flow Cytometry, Recombinant, Staining, Selection, Antibody Labeling, Labeling, Isolation, Sample Prep, Luminex, Quantitative Proteomics, Generated, Gene Expression, Clone Assay, Marker, Expressing, Mass Spectrometry, Derivative Assay, RNA Sequencing, Sequencing, Illumina Sequencing, Software, Variant Assay